The goal of this proposal is to develop microfluidic devices for effective single-cell analysis in biomedical laboratories without the requirement of specialized expertise. The lack of ability to perform biochemical and molecular characterization at the single-cell level is a critical obstacle in cancer research and diagnosis. We have developed microfluidic devices for efficient large-scale single-cell analysis to overcome this obstacle. The technology has broad applications in cancer research and diagnosis for identification, enumeration and characterization of the rare cells including 1) circulating tumor cells (CTC) in occult metastases;2)T-regulatory cells, 3) tumor-specific T-cytotoxic cells;4) cancer stem cells;5) malignant cells captured by laser capture microdissection (LCM);and 6) suspicious malignant cells in needle biopsy. Isolation methods have been developed to capture rare cancer cells from blood and other tissues for cancer diagnosis. However, there is still a lack of robust methods to perform biochemical and molecular characterization on these captured cells at the single-cell levels. Here, we propose to develop an inexpensive and integrated microfluidic device that can simultaneously profile 100 single-cells within 3 hours. The proposed device can be fabricated and used in general laboratories without the need of specialized expertise, and has a 5 fold higher mRNA-to-cDNA efficiency than current bulk assays. With our microfluidic devices, multiple biochemical analyses can now be performed at the single-cell levels with a limited number of target cells such as the rare cancer cells listed above. We propose to optimize our devices for single-cell biochemical characterization of CTC captured by our membrane filters. Characterization of these captured CTC is likely to lead to the identification of molecular therapeutic targets. After developing and optimizing the device with this R21 proposal, a R33 proposal will be submitted to apply this device for molecular characterization of CTC captured from bloods of cancer patients at the single-cell level.